Nematode parasites infect approximately two billion people worldwide. Ascaris, a genus of clade III nematode parasites, is a gastrointestinal roundworm that infects humans, pigs, and other animals worldwide (Taylor et al., Emerging Infectious Diseases 22(2): 339-340 (2016)). Ascaris has been estimated to cause more than 1.2 billion human infections (de Silva et al., Trends Parasitology 19(12): 547-551 (2003)).
With no effective vaccines and inadequate sanitation in many countries, the control of Ascaris infection mainly relies on the limited number of available anthelmintic drugs. Some of these drugs act on nicotinic acetylcholine receptors (nAChRs). The nAChRs are pentameric ligand-gated ion channels involved in synaptic transmission in the nervous systems of vertebrates and invertebrates (Taly et al., Nat Rev Drug Discov 8(9): 733-750 (2009)). The receptors also serve a paracrine function in non-excitable tissues (Proskocil et al., Endocrinology 145: 2498-2506 (2004)). The nAChRs are activated by the ligands acetylcholine (ACh), nicotine, and structurally related derivatives, which leads to the opening of their transmembrane ion-channels and flux of sodium, potassium, and sometimes calcium ions, across the membrane. The agonist-binding sites of nAChRs have been well-studied using a combination of photolabeling, mutagenesis, and electrophysiology (Arias, Neurochem Int 36(7): 595-645 (2000)).
Acetylcholine binding proteins (AChBPs) are homologs of the extracellular agonist-binding site domain of nAChRs and share 20-24% sequence identity with the extracellular domain of AChRs (Blum et al., PNAS USA 107(30): 13206-13211 (2010)). Co-crystallization of invertebrate acetylcholine binding proteins (AChBPs) and cholinergic ligands has increased understanding of ligand interactions with the receptors (Rucktooa et al., Biochemical pharmacology 78(7): 777-787 (2009); and Sixma and Smit, Annual Review of Biophysics and Biomolecular Structure 32: 311-334 (2003)). The agonist-binding site of nAChRs is at the interface between the principal subunit (an α subunit with vicinal cysteines) and the adjacent complementary subunit (without vicinal cysteines) in the extracellular domain (ECD). Five aromatic amino acids in the agonist-binding site are highly conserved in nAChRs and contribute to the cation-π interactions with the cationic nitrogen in the nAChR agonists (Dougherty, Accounts of Chemical Research 46(4): 885-893 (2013)). Another feature of nAChR agonists is the hydrogen bond acceptor, which is about 4-6 Å from the cationic nitrogen. Based on the high-resolution structures of AChBPs, the hydrogen bond acceptor in the agonist is stabilized by a water molecule, which interacts with the carbonyl or the amide backbones of two less conserved residues on loop E of the complementary subunit through three hydrogen bonding interactions (Van Arnam and Dougherty (2014), supra).
The nicotinic acetylcholine receptor subtype 16 from Ascaris suum (Asu-ACR-16) is a homopentameric receptor, which resembles vertebrate α7 nAChRs (Mongan et al., Protein science: a publication of the Protein Society 11(5): 1162-1171 (2002)). Asu-ACR-16 is widely distributed in A. suum tissues but its physiological function remains to be determined (Abongwa et al., British Journal of Pharmacology (2016) DOI: 10.1111/bph.13524; and Zheng et al., Int'l J for Parasitology: Drugs and Drug Resistance 6(1): 60-73 (2016)). Asu-ACR-16 is pharmacologically different from its host α7 nAChR and may be exploited as an anthelmintic drug target to counter resistance to cholinergic anthelmintics directed at other pharmacological types of nAChR (Holden-Dye et al., Parasitology Int'l 62(6): 606-615 (2013); and Zheng et al. (2016), supra).
The agonist-binding site of the Asu-ACR-16 can be predicted by homology modeling using a human α7 nAChR chimera as a structural template. The chimera shares 38% identity and 73% sequence similarity with Asu-ACR-16. Five conserved aromatic residues and two hydrogen bond-interacting residues have orientations very close to corresponding residues in other nAChRs (Zheng et al. (2016), supra).
The Asu-ACR-16 is sensitive to six nicotinic agonists, namely nicotine, acetylcholine (Ach), cytisine, 3-bromocytisine, epibatidine, and dimethyl-4-phenylpiperazinium iodide (DMPP), and insensitive to other cholinergic anthelmintic agonists (Abongwa et al. (2016), supra). All six agonists share the nicotinic pharmacophore, which is a cationic nitrogen separated by ˜5 Å from a hydrogen bond acceptor.
There is an urgent need for new drugs, given that resistance in various nematodes has been reported following frequent use of anthelmintic drugs (Garcia et al., Anais da Academia Brasileira de Ciencias 88(1): 397-402 (2016)). It is an object of the present disclosure to provide novel nicotine alkaloids, in particular nicotine alkaloids that are effective agonists of nAchRs in nematode parasites. This and other objects, and advantages, of the present disclosure will become apparent from the detailed description provided herein. The nicotine alkaloids provided herein have the potential to circumvent drug resistance that has arisen following repeated treatment of nematode parasites with other classes of anthelmintic drugs and are a useful lead for anthelmintic drug development.